Ciz1, a Novel DNA-Binding Coactivator of the Estrogen Receptor A, Confers Hypersensitivity to Estrogen Action Petra Den Hollander,1,2 Suresh K

Research Article

Ciz1, a Novel DNA-Binding Coactivator of the Estrogen Receptor A, Confers Hypersensitivity to Estrogen Action Petra den Hollander,1,2 Suresh K. Rayala,1 Dawn Coverley,4 and Rakesh Kumar1,2,3

1Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center; 2The University of Texas Graduate School of Biomedical Sciences at Houston; 3Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas; and 4Lister Institute of Preventive Medicine, University of York, York, United Kingdom

Abstract a number of regulatory cofactors, including chromatin-remodeling The transcriptional activity of the estrogen receptor (ER) complexes, coactivators, and corepressors, that are recruited to is affected by regulatory cofactors, including chromatin- target gene promoters through protein-protein interactions with remodeling complexes, coactivators, and corepressors. ER and function as linker molecules between the DNA, DNA- binding proteins, and DNA-modifying enzymes. Coregulators are recruited to target gene promoters through protein-protein interactions with ER and function as linker Tumor progression involves the gradual transition of the normal molecules between the DNA, DNA-binding proteins, and DNA- phenotype to the malignant phenotype through a series of modifying enzymes. We recently showed that Cip-interacting cumulative cellular, genetic, and epigenetic alterations. During zinc finger protein 1 (Ciz1) participates in the regulation of the tumorigenesis, the cellular machineries responsible for the cell cycle in estrogen-stimulated breast cancer cells. Despite proliferative, migratory, and invasive properties become deregu- the emerging significance of Ciz1 in the biology of breast lated, enabling altered cells to grow, survive, and metastasize to cancer cells, regulation of endogenous Ciz1 in hormone- distant organs. The progression of hormone-responsive cancers is responsive cancer cells remains unknown. To shed light on also characterized by the deregulation of the cell cycle and the role of Ciz1 in breast tumorigenesis, we defined the cytoskeleton signaling. Furthermore, the development of breast regulation of Ciz1 by the ER pathway and found that Ciz1 is cancer is profoundly influenced by the stimulatory action of an estrogen-responsive gene. We also discovered that Ciz1 estrogen. Accordingly, studies on the effects of estrogen on cell protein, a DNA-binding factor, coregulates ER by enhancing proliferation and differentiation have focused primarily on the ER transactivation activity by promoting the recruitment of role of estrogen and its receptors in controlling the entry into, the ER complex to the target gene chromatin. In addition, we progression through, and exit from the G1 phase of the cell cycle. found that Ciz1 overexpression confers estrogen hypersensi- Some of the regulatory components involved in the G1-S tivity to breast cancer cells and promotes the growth rate, transition are the cyclin D1-cdk4 and cyclin E-cdk2 complexes, anchorage independency, and tumorigenic properties of breast which phosphorylate substrates such as pRB. Estrogen induces a cancer cells. These findings revealed the inherent role of Ciz1, significant increase in the phosphorylation of pRB (3–5), thereby a novel DNA binding and ER coactivator, in amplifying allowing the initiation of DNA synthesis (6). Treatment with estrogenic responses and promoting breast cancer tumorigen- antiestrogenic agents triggers a decline in cyclin D1 (7), which can esis. (Cancer Res 2006; 66(22): 11021-9) be up-regulated again by estrogen treatment owing to increased cyclin D1-cdk4 association and cdk4 activity (8). Another well- Introduction recognized estrogen target is the transcription factor c-Myc, which controls a number of cellular pathways. Rapid and direct The development of human breast cancer is promoted by regulation of c-Myc by estrogen places c-Myc induction among estrogen stimulation of mammary epithelial cell growth. The the earliest detectable transcriptional responses to estrogen. biological effects of estrogen are mediated by its binding to the Furthermore, functionally inactivating c-Myc with the use of structurally and functionally distinct estrogen receptors (ER) a and h a antisense oligonucleotides inhibits estrogen-stimulated breast .ER is the major ER in the human mammary epithelium. cancer cell proliferation (9). Estrogen triggers the stimulation of ERa, which interacts directly A large body of previous work has provided detailed insight with estrogen response elements (ERE) in target gene promoters to about the roles of cell cycle components or coregulatory proteins in induce gene transcription (1, 2). ERs also regulate the transcription transcriptional stimulation by estrogen in breast cancer cells. of ER target genes through nonclassic response sites involving However, the specific participation of coactivators in cell cycle protein-protein interactions with transcription factors such as Sp1, a regulation remains poorly defined. An earlier study from this c-Jun, and activator protein-1 (2). Ligand-activated ER undergoes laboratory revealed a coactivator function of dynein light chain 1 a conformational change that initiates a series of events leading to (DLC1) in targeting the ER to the chromatin of estrogen-inducible the formation and recruitment of multiprotein complexes to the genes in breast cancer cells (10). More recently, we showed that target gene chromatin and, consequently, the transcription of DLC1 interacts with a novel protein, Cip-interacting zinc finger ER-regulated genes. The transcriptional activity of ER is affected by protein 1 (Ciz1), and participates in the regulation of cell cycle by increasing cdk2 kinase activity and inducing the G1-Stransition (11, 12). Despite the emerging significance of Ciz1 in the biology Requests for reprints: Rakesh Kumar, Molecular and Cellular Oncology, The of breast cancer cells, how endogenous Ciz1 is regulated in University of Texas M.D. Anderson Cancer Center, Houston, TX 77030. E-mail: hormone-responsive cancer cells is unknown. To shed light on the [email protected]. I2006 American Association for Cancer Research. role of Ciz1 in breast tumorigenesis, here we studied the gene doi:10.1158/0008-5472.CAN-06-2336 regulation of Ciz1 by the ER pathway and found that Ciz1 is an www.aacrjournals.org 11021 Cancer Res 2006; 66: (22). November 15, 2006

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Cancer Research estrogen-responsive gene. We discovered that Ciz1 protein, a DNA- 5¶-AGGATTTGCTGATAGACAGAGACGAC-3¶. For the pS2 upstream primers, binding factor, coregulates ER by enhancing ER transactivation the sequences were 5¶-CTCCCTCTTCAGGCCTCTCT-3¶ and 5¶-TTCCCTGGT- ¶ ¶ ¶ activity and recruitment to target gene chromatin, and contributes GTTGTCAAGTG-3 . For ERE1 in Ciz1,5-GGAGCCCTTCAACAGGAGAT-3 ¶ ¶ ¶ to hypersensitivity of breast cancer cells to estrogen. In addition, and 5 -CATCCTGCTGGAATGGAAGT-3 . For ERE2 in Ciz1, 5 -TTCAATAC- CGCAACCCTCTC-3¶ and 5¶-GCTGTGTCTGCGGAGGAG-3¶. overexpression of Ciz1 promoted the growth-rate, anchorage- Reporter gene assay. For reporter gene transient transfections, cells independence and tumorigenesis of breast cancer cells. were cultured for 48 hours in minimal essential medium, without phenol red, containing 5% charcoal-stripped serum. Next, 200 ng ERE-luciferase reporter constructs were cotransfected with 500 ng cytomegalovirus (CMV) Materials and Methods or Ciz1 plasmid using the Fugene-6 reagent according to the protocol of Cell cultures and reagents. Cell lines stably overexpressing Ciz1 were the manufacturer (Roche, Indianapolis, IN). In the case of assaying the generated by transfecting ZR-75 human breast ductal carcinoma cells Ciz1 promoter, 100 ng promoter construct were transfected with 500 ng (American Type Culture Collection, Manassas, VA) with pcDNA-T7-Ciz1. pcDNA or ER. Twenty-four hours later, the cells were treated with estrogen À After 48 hours, the cells were selected in medium containing G418 (10 9 mol/L) for 24 hours. The cells were then lysed with passive lysis buffer, (500 Ag/mL). ZR-75 cells were maintained in RPMI supplemented with 10% and the luciferase assay was done using a kit (Promega). h-Galactosidase fetal bovine serum (FBS). MCF-7, T47D, MDA231 (human breast ductal activity was used to normalize the transfection. Each transfection was done carcinoma cells), and HeLa (human cervical carcinoma) cells were in six-well plates in triplicate. maintained in DMEM/F12 supplemented with 10% FBS. Estrogen was GST pull-down assay. In vitro transcription and translation of the Ciz1 purchased from Sigma-Aldrich (St. Louis, MO). Charcoal-stripped serum and ER proteins were done using the TNT transcription/translation system was purchased from Gemini Bio-Products (Woodland, CA). Antibodies (Promega) in the presence of [35S]methionine. The reaction mixture was against cyclin D1 were from Neomarkers (Fremont, CA), those against diluted 10 times with NP40 lysis buffer (25 mmol/L Tris, NaCl 50 mmol/L, vinculin were from Sigma-Aldrich, those against ER were from Chemicon and 1% NP40). The GST pull-down assays were done by incubating equal International (Temecula, CA), those against T7 tag were from Novagen (San amounts of GST, GST-Ciz1-domain, GST-ER, or GST-ER deletion proteins Diego, CA), those against proliferating cell nuclear antigen (PCNA) were immobilized on GST beads (Amersham Pharmacia Biotech, Piscataway, NJ) from Genetex (San Antonio, TX), and those against Ki-67 were from with in vitro-translated recombinant protein. Bound proteins were isolated Invitrogen (Carlsbad, CA). The anti-Ciz1 antibody used here has been by incubating the mixture for 2 hours at 4jC and then washed five times characterized previously (13). with NP40 lysis buffer. The proteins were eluted with a 2Â SDS buffer, Plasmid construction. Glutathione S-transferase (GST)-Ciz1 full-length separated by SDS-PAGE, and visualized by autoradiography. and deletion constructs were generated as described earlier (11). To Cell extracts, immunoblotting, and immunoprecipitation. To generate an expression vector containing Myc-tagged Ciz1 (Myc-Ciz1) or a prepare the cell extracts, cells were washed thrice with PBSand lysed in Ciz1 expression vector with T7 tag (T7-Ciz1), Ciz1 cDNA was amplified by buffer [50 mmol/L Tris-HCl (pH 7.5), 120 mmol/L NaCl, 1% Triton X-100, PCR and cloned into pCMV-Myc vector (Clontech, Palo Alto, CA) or 1Â protease inhibitor mixture (Roche), and 1 mmol/L sodium vanadate] for pcDNA3.1 vector (Invitrogen), respectively. Expression of Myc-Ciz1 and 30 minutes on ice. Cell lysates containing equal amounts of protein were T7-Ciz1 were verified by immunofluorescence and Western blotting with then resolved on an SDS-polyacrylamide gel. The proteins were transferred Myc and T7 epitope monoclonal antibody (mAb), respectively. Luciferase to nitrocellulose, probed with the appropriate antibodies, and detected construct for the Ciz1 ERE was generated by amplifying the ERE and using an enhanced chemiluminescence method. The cell lysates for flanking regions by PCR and cloning them into pGL3-luciferase vector. immunoprecipitation were prepared using NP40 lysis buffer [50 mmol/L ERE-luciferase and pS2-luciferase were described previously (10). Tris-HCl (pH 7.5), 100 mmol/L NaCl, 0.5% NP40, 1Âprotease inhibitor RNA extraction, reverse transcription-PCR, and real-time quantita- cocktail, and 1 mmol/L sodium vanadate]. Immunoprecipitation was tive PCR. RNA was extracted with Trizol reagent (Invitrogen) according to done for 4 hours at 4jC using 1 Ag of antibody per milligram of protein. The the instructions of the manufacturer. Reverse transcription-PCR (RT-PCR) anti-Ciz1 antibody used in these experiments has been characterized was done using the Access RT-PCR kit from Promega (Madison, WI) and previously (13). 100 ng RNA according to the protocol of the manufacturer. Real-time PCR Electrophoretic mobility shift assay. Electrophoretic mobility shift was done by generating cDNA using the First Strand cDNA synthesis kit assay with radiolabeled probe was done with commercial double-stranded (Invitrogen). The Taqman assay with iQ Supermix (Bio-Rad, Hercules, CA) ERE consensus sequence probes purchased from Santa Cruz Biotechnology was used for quantitative analysis of Ciz1 expression and normalized with (Santa Cruz, CA). The probes were end-labeled using T4-polynucleotide actin expression using a 6-FAM-TAMRA-labeled probe (Applied Biosystems, kinase (Invitrogen) and 32P-labeled ATP (Perkin-Elmer, Wellesley, MA). Foster City, CA). Primers and probes used for Ciz1 were forward 5¶- Binding to ERE was visualized by running a 5% acrylamide gel, drying the ACATATCCACAGGTCCACACAC-3¶, reverse 5¶-CTGCTCATGGGTC- gel, and visualizing the results by autoradiography. TGCTCTG-3¶, probe (6-FAM) 5¶-CACAGCCAAGCGTCCAGCCACAGG-3¶ Cell growth and soft-agar experiments. For the cell growth experi- (TAMRA), and those used for actin were forward 5¶-TGACTGACTACCT- ments, equal numbers of ZR-75/Ciz1 and ZR-75/pcDNA cells were plated in CATGAAGATCC-3¶, reverse 5¶-CCTTAATGTCACGCACGATTTCC-3¶,probe triplicate in 24-well plates for 48 hours in phenol red–free DMEM (6-FAM) 5¶-CGGCTACAGCTTCACCACCACGGC-3¶(TAMRA). All experiments supplemented with 5% charcoal-stripped serum. The cells were then À were done in duplicate. The relative induction of Ciz1 mRNA was then treated with estrogen (10 9 mol/L) for 6 days, and their growth rate was calculated using the comparative CT method. measured on days 0, 2, 4, and 6 with a Coulter Counter (Beckman Coulter, Chromatin immunoprecipitation assay. Chromatin immunoprecipi- Fullerton, CA). tation (ChIP) assays were done as described previously (14). Equal amounts Colony growth assays were done as described previously (14). Briefly, 1 of DNA were used for each sample. For input DNA, 10% of the chromatin mL solution of 0.5% noble agar in modified essential medium supplemented solution was put aside before immunoprecipitation. Chromatin solutions with 5% charcoal-stripped serum was layered onto 30 Â 10-mm tissue were immunoprecipitated at 4jC overnight. PCR analysis was done with culture plates. A total of 1 Â 104 cells was mixed with 1 mL of 0.3% agar primers for the Ciz1 gene (ERE1 +6,213 and ERE2 +13,111) and for pS2 gene solution prepared in a similar manner and layered on top of the 0.5% j (pS2 promoter primers) À463 to À159 or 1 kb upstream of this element to agar layer. Plates were incubated at 37 Cin5%CO2 for 21 days. When À serve as a negative control (pS2 upstream primers) À1,953 to À1,651; 3 AL indicated, some cultures were treated with estradiol (E2; 10 9 mol/L) or the À of sample DNA; and 1 AL of input DNA. PCR was restricted to 30 cycles. antiestrogenic agent ICI-182780 (10 8 mol/L). The experiment was done PCR products (228, 172, 304, and 302 bp, respectively) were resolved on a 2% in triplicate. agarose gel and visualized with ethidium bromide. The sequences of the pS2 Mouse xenograft studies. For the tumorigenesis studies, 3 days after promoter primers were 5¶-GAATTAGCTTAGGCCTAGACGGAATG-3¶ and implantation of a 60-day release estrogen pellet, 4-week-old ovariectomized

Cancer Res 2006; 66: (22). November 15, 2006 11022 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Ciz1 Is an ERa Coactivator female athymic nude mice (Charles River, Wilmington, MA) were bilaterally The cells were incubated first with primary antibodies and then with injected with 5 Â 106 cells of either ZR-75/pcDNA or ZR-75/Ciz1 clones secondary antibodies conjugated with Alexa-546 (red) and Alexa-488 (green; into the mammary fat pads, as previously described (15). Tumors were both from Molecular Probes, Eugene, OR). Topro 3 (blue; Molecular Probes) allowed to grow for 5 weeks, and tumor size was measured every 15 days. was used to counterstain the DNA. Cells treated with only the secondary The animals were divided into four groups. Group 1 mice were injected antibodies served as the controls. Confocal scanning analysis was done with ZR-75/pcDNA cells. In group 2 mice, estrogen pellets were implanted using an FV300 laser-scanning confocal microscope (Olympus, Olympus 3 days before the ZR-75/pcDNA cells were injected. The mice in groups America, Inc., Center Valley, PA). Each slide was examined for each stain at 3 and 4 were treated similarly, but were injected with ZR-75/Ciz1 cells. three excitation wavelengths (488, 546, and 633 nm), and the data were All animal procedures were done in compliance with the Institute Animal compared pixel by pixel. Colocalization was indicated by the development Care and Use Committee and the NIH Policy on Humane Care and Use of a yellow color. of Laboratory Animals. Immunohistochemical analysis. For immunohistochemical detection Results of Ki-67, PCNA, and cyclin D1, xenograft sections were deparaffinized with Ciz1 xylene; rehydrated using graded ethanol; incubated in 0.3% H2O2 and is an estrogen-inducible gene. To investigate the role of methanol for 30 minutes to inactivate endogenous peroxidase; boiled for Ciz1 in the estrogen signaling pathway in breast cancer cells, we 15 (Ki-67), 20 (PCNA), and 30 (cyclin D1) minutes in 0.01 mol/L citrate first searched for the potential presence of ERE in the putative Ciz1 buffer; and cooled for 30 minutes at room temperature to expose antigenic promoter both upstream and downstream of the transcriptional epitopes. The sections were incubated with 2% normal goat serum in 1% start site of Ciz1 (Genbank accession no. NM_012127). The fact that bovine serum albumin and PBSfor 30 minutes and then with anti-Ki-67, we found multiple half-EREs and two palindromic EREs in the first j anti-PCNA, and anti-cyclin D1 antibodies and incubated overnight at 4 C. intron of the Ciz1 gene (Fig. 1A, top) raised the possibility that Ciz1 The sections were washed thrice with 0.05% Tween in PBSfor 10 minutes, is an estrogen-inducible gene. To explore this hypothesis, we incubated with secondary antibody, developed with 3,3¶-diaminobenzidine- treated MCF-7 and ZR-75 breast cancer cells with estrogen and H2O2, and counterstained with Mayer’s hematoxylin. Negative controls were done by replacing the primary antibody with the corresponding IgG. found that it rapidly induced the level of Ciz1 mRNA in both cell Immunofluorescence and confocal studies. The cellular localization lines (Fig. 1A, bottom). To validate these finding in a quanititative of different proteins was determined by indirect immunofluorescence. manner, we did real-time PCR for a panel of estrogen-sensitive and Briefly, ZR-75 cells were grown on glass coverslips and fixed with methanol. ER-negative breast cancer cell lines. Estrogen induced Ciz1 in the

Figure 1. Ciz1 is an estrogen-inducible gene. A, top, map of ERE in Ciz1 gene chromatin. A, bottom, B, and C, Ciz1 mRNA induction by estrogen. A, bottom, MCF-7 and ZR-75 cells were treated with E2 (10À9 mol/L) for 6, 12, or 24 hours. B, ZR-75, T47D, HeLa, and MDA231 cells were treated with E2 (10À9 mol/L) for 24 hours. C, induction of Ciz1 mRNA can be blocked by ICI-182780 and is caused by enhanced transcription of the Ciz1 gene. MCF-7 cells were treated with E2 (10À9 mol/L) for 6, 12, or 24 hours. Pretreatment was 1 hour with ICI-182780 and 4 hours with actinomycin D. For (B) and (C), the relative induction of Ciz1 mRNA was calculated using the comparative CT method. All experiments were done in duplicate. D, induction of Ciz1 protein levels by estrogen. MCF-7 and ZR-75 cells were treated with E2 (10À9 mol/L) for 24 hours.

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ER-positive cells, but not in the ER-negative cells (Fig. 1B). The between the first and second zinc finger motifs (Fig. 3A). We next specificity of this estrogen induction of Ciz1 mRNA in an carried out a series of GST pull-down studies using in vitro– ER-dependent manner was confirmed by using the pure anti- translated ER and Ciz1-deletion GST-fusion proteins. We found estrogenic agent ICI-182780 (Fig. 1C, compare lanes 4 and 5). The no evidence of ER interaction with the LXXLL motif–containing observed increase in the levels of Ciz1 mRNA was due to newly region of Ciz1. Rather, Ciz1 used a region containing the second synthesized Ciz1 mRNA, because pretreatment of the cells with glutamine-rich region at the NH2 terminus of the protein to actinomycin D, a transcriptional inhibitor, prevented the ability of interact with the ER (Fig. 3A). Because Ciz1 did not interact with estrogen to induce the levels of Ciz1 mRNA (Fig. 1C). Estrogen ER via its NR box, we expected to find that Ciz1 interacts with a stimulation of MCF-7 and ZR-75 cells was also accompanied by region or regions other than the AF2 domain of ER. Indeed, Ciz1 increased levels of Ciz1 protein (Fig. 1D). did not interact through its AF2 domain of ER; instead, it bound to To define the molecular basis of estrogen stimulation of Ciz1 the DNA-binding domain of ER (Fig. 3B). expression, we focused on the palindromic EREs present in the We next examined the physiologic interaction between Ciz1 and Ciz1 gene chromatin and explored whether ER is recruited to ER in breast cancer cells. ZR-75 breast cancer cells were stimulated these elements. To this end, we carried out ChIP studies to with estrogen, the cell lysates were immunoprecipitated with an examine the potential recruitment of ER to two elements (ERE1 anti-Ciz1 antibody, and the precipitated material was immuno- +6,213 bases and ERE2 +13,111 bases from the transcriptional blotted with antibodies against ER and Ciz1. We found that start site) in the first intron of Ciz1. ER was indeed recruited onto estrogen stimulation enhances the physical interaction between both elements and with a similar cyclic pattern, first at 15 minutes ER and Ciz1 dramatically (Fig. 3C). We also detected distinct and again at 60 minutes (Fig. 2A). An earlier study showed that colocalization of ER and Ciz1 in the nuclear compartment by the first recruitment of ER is nonfunctional but at the second scanning confocal microscopy (Fig. 3D). These results suggested recruitment, ER is recruited as a more potential inducer of that ER and Ciz1 interact in a ligand-dependent manner in a transcription and induces gene expression (16). To show the physiologic setting. specificity of the association of ER with the Ciz1 chromatin, we Ciz1 is a coactivator of ERA. One of the primary cellular did a PCR of a part of the pS2 promoter known not to interact actions of ER is the induction of gene transcription via interaction with ER (Fig. 2A, right). We next did a double ChIP of RNA with the ERE in the target genes. Because we found that Ciz1 polymerase II and ER and assessed its association with Ciz1 physically interacts with ER in vivo and that Ciz1 is predominantly chromatin upon estrogen treatment. We found that, indeed, RNA a nuclear protein, we speculated that Ciz1 is associated with the ER polymerase II and ER are associated with ERE1 and ERE2 regions target gene chromatin. Using ChIP–based assays and ZR-75 cells of the Ciz1 gene chromatin in a estrogen-dependent manner stably expressing T7-Ciz1, we found that T7-Ciz1 interacted with (Fig. 2B). We cloned the Ciz1 gene fragment that contains the ERE the pS2 chromatin, which is an estrogen-inducible gene (Fig. 4A, into a pGL2-luciferase reporter system and showed that estrogen left). We also noticed a potentiating effect of T7-Ciz1 expression stimulation of breast cancer cells was accompanied by increased on the amount of ER recruited to the pS2 gene, and this effect transcriptional activity of Ciz1 (Fig. 2C). Taken together, these was further enhanced on estrogen stimulation (Fig. 4A, left). The findings suggested that Ciz1 is an estrogen-inducible gene specificity of the binding of Ciz1 to the pS2 chromatin was product. determined by using a primer set 1 kb upstream of the ERE in pS2, Ciz1 interacts with ER. Earlier studies have shown that many which served as a negative control (Fig. 4A, middle). To show the estrogen-inducible gene products, such as PELP1 and DLC1, existence of ER and Ciz1 within the same complex, we did a double interact with ER and thus are recruited to the target gene ChIP assay involving the first ChIP with an anti-T7 mAb to chromatin as a complex with ER (10, 17). For this reason, we precipitated T7-Ciz1 and a second ChIP with an ER-mAb. We searched the protein sequence of Ciz1 for the known nuclear found that, indeed, T7-Ciz1/ER complex is effectively recruited to receptor–binding motif LXXLL and discovered one such motif the pS2 chromatin (Fig. 4A, right).

Figure 2. ER is recruited to the ERE in Ciz1 chromatin and induces gene expression. A, ER recruitment to the ERE in Ciz1 gene. MCF-7 cells were treated with estrogen (10À9 mol/L) for 15, 30, 45, or 60 minutes before ER was immunoprecipitated with the associated chromatin. PCR for Ciz1 was done. A, right, PCR for pS2 upstream of ERE was done as negative control. B, Pol II recruitment to the Ciz1 gene. MCF-7 cells were treated with estrogen (10À9 mol/L) for 60 minutes before ER was immunoprecipitated with the associated chromatin and after this Pol II using a Pol II antibody. PCR for Ciz1 was done. C, induction of Ciz1 promoter luciferase by ER. MCF-7 cells were transfected with pcDNA or ER and with Ciz1 promoter constructs. Cells were deprived of E2 for 48 hours before E2 (10À9 mol/L) was added for 24 hours.

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Figure 3. Ciz1 binds ER in vivo and directly in vitro. A, ER interacts with the second glutamine-rich region of Ciz1. GST pull-down assays showthe association of GST-Ciz1 deletion constructs with in vitro–translated 35S-ER. Arrows, deletion protein bands. B, Ciz1 interacts with the DNA-binding domain of ER. GST pull-down assays show the association of GST-ER deletion constructs with in vitro–translated 35S-Ciz1. Arrows, deletion protein bands. C, Ciz1-ER interaction is enhanced after estrogen stimulation. ZR-75 cells were treated with E2 (10À9 mol/L) for 1 hour before Ciz1 was immunoprecipitated. D, Ciz1 (green) and ER (red) localize in the nucleus. Yellow, colocalization.

Because we found that Ciz1 associated with the ERE-containing regulation of cyclin D1 may contribute to the Ciz1-mediated pS2 chromatin and potentiated the recruitment of ligand-activated stimulation of DNA replication reported previously (13). These ER to that chromatin, we hypothesized that Ciz1 might act as a results supported the notion that Ciz1 acts as a coactivator of ER coactivator for ER. To test this hypothesis, we did a luciferase assay and induces hypersensitivity to estrogen in breast cancer cells. using a reporter construct containing EREs and found that Ciz1 Ciz1 promotes tumorigenic phenotypes in breast cancer acts as a coactivator for ER and increased its transactivation cells. We explored the functional consequence of the over- activity under both basal and ligand-induced conditions (Fig. 4B, expression of the newly identified coregulator Ciz1. Ciz1 over- left). Consistent with these results, selective knockdown of expression enhanced the growth rate of ZR-75 cells under both endogenous Ciz1 by using Ciz1-specific small interfering RNA estrogen-depleted and estrogen-supplemented conditions (Fig. 5A). reduced the transactivation activity of ER (Fig. 4B, middle), When we evaluated the specificity of those results, we observed suggesting an inherent role of Ciz1 in optimal ER responsiveness. that the addition of the pure antiestrogenic agent ICI-184280 To further validate our discovery that the coactivator function of resulted in complete abrogation of the growth-promoting activity Ciz1 is due to direct binding of the Ciz1-ER complex to the ERE in a of Ciz1-overexpressing cells (Fig. 5B). This finding strengthened the physiologically relevant endogenous promoter, we used a more idea that Ciz1 overexpression induces hypersensitivity to estrogen physiologically relevant endogenous promoter of pS2 and were still in breast cancer cells. In addition, Ciz1-overexpressing cells grew able to observe induction after Ciz1 transfection (Fig. 4B, right). in an anchorage-independent manner (Fig. 5C) that could be We then carried out gel-shift studies using an oligonucleotide completely reverted to the control level on addition of ICI-184280 containing ERE by using the nuclear extracts from T7-Ciz1– (Fig. 5D). Together, these observations suggested that Ciz1 transfected cells. As we expected, a distinct ER-Ciz1 complex enhances the tumorigenic phenotype of breast cancer cells in an formed on the ERE itself that could be supershifted by antibody ER-dependent manner. against ER or Ciz1 (Fig. 4C). The above findings suggested that Ciz1-overexpressing cells induce tumor formation in nude Ciz1 is recruited to the native target gene chromatin. We also mice. In general, tumor formation by ER-positive cells, such as showed that Ciz1 overexpression promoted the expression of cyclin ZR-75 cells in immunocompromised mice, depends on continuous D1 (Fig. 4D, top). estrogenic stimulation by estrogen pellets (15). To examine the The fact that Ciz1 expression potentiated the estrogen effect of Ciz1 overexpression on the tumor-forming ability of breast stimulation of ER transactivation activity, ER recruitment to the cancer cells, we examined the ability of ZR-75/pcDNA and ZR-75/ target gene chromatin, and expression of ER target gene cyclin D1 Ciz1 cells to form tumors in a xenograft model involving suggested that Ciz1 confers estrogen hypersensitivity to breast implantation of estrogen pellets in ovariectomized female nude cancer cells. To test this notion at a biochemical level, we mice. ZR-75 cells are known to form tumors in estrogen-treated stimulated the Ciz1-overexpressing and control cells with a serially mice, which was also observed in our study. Ciz1 overexpression À À À diluted dose of estrogen over 6 logs (10 9,10 11, and 10 13 mol/L in these ZR-75 cells induced a significantly higher amount of E2) and evaluated the expression of cyclin D1 as an indicator of tumors in the mice with estrogen pellet (m2; P = 0.01; Fig. 6A). Ciz1 estrogen sensitivity. Ciz1 expression conferred hypersensitivity to overexpression conferred tumor-forming ability to ZR-75 cells the responsiveness of breast cancer cells to estrogen: Cyclin D1 was even without exogenous estrogen treatment, suggesting that induced by a very low dose of estrogen in the Ciz1-overexpressing deregulation of Ciz1 might be sufficient to promote estrogen cells but not in the control cells (Fig. 4D, bottom). This noted up- sensitivity and tumorigenic phenotypes. www.aacrjournals.org 11025 Cancer Res 2006; 66: (22). November 15, 2006

As we expected, the tumors formed by ZR-75/Ciz1 cells were of these cells. They also suggest that the reported ability of Ciz1 to larger than the tumors formed by estrogen-stimulated ZR-75/ promote DNA replication may be partly mediated via up- pcDNA cells (Fig. 6A, graph). Thus, Ciz1 seems to have a role in regulation of cyclin D1. promoting ligand-induced tumorigenic activity of breast cancer cells. To determine whether the larger tumor size was caused by increased cell proliferation, we did immunohistochemical analysis Discussion of PCNA and Ki-67, and found higher staining levels in tumors We found that Ciz1 is an estrogen-inducible gene product that with Ciz1 overexpression, which translates into a higher proli- facilitates ER recruitment to ER target gene chromatin and ER ferative index (Fig. 6B). To determine whether Ciz1-expressing transactivation activity (Fig. 6D). The Ciz1-mediated increased ER tumors also exhibit increased levels of ER target genes, such as transactivation activity was functional, because it was accompa- cyclin D1, we carried out immunohistochemical analysis of cyclin nied by increased protein expression of ER target genes, enhanced D1 in the tumor samples from all treatment groups. Consistent cell proliferation and soft-agar colony formation, and increased with the results from our tissue culture studies, tumors that ability of Ciz1-expressing cells to form tumors after estrogen formed in the ZR-75/Ciz1 group, stimulated by estrogen, exhibited stimulation in mice. Another interesting observation was the clearly enhanced levels of cyclin D1 (Fig. 6C). These findings complete blockage by the antiestrogenic agent ICI-184280 of the suggested a role of Ciz1 in promoting the estrogenic stimulation observed growth-promoting advantage of Ciz1-expressing breast of breast cancer cells, thereby leading to the tumor-forming ability cancer cells, because the proliferation, and anchorage-independent

Figure 4. Ciz1 is a coactivator of ER. A, Ciz1 is recruited to the ER target gene chromatin and enhances ER recruitment. ZR-75/pcDNA and ZR-75/Ciz1 cells were treated with E2 (10À9 mol/L) for 1 hour before cross-linking. Lysates were immunoprecipitated with anti-T7-Ciz1 or anti-ER antibody. PCR was done for ERE in the pS2 chromatin and 1 kb upstream as a negative control. B, Ciz1 affects ER transactivation activity. Left, Ciz1 overexpression increases ER transactivation activity. MCF-7 cells were transfected with CMV, Ciz1, and ERE-luciferase for 24 hours and treated with E2 (10À9 mol/L) for 24 hours, and then ERE-luciferase activity was measured. Middle, Ciz1 knockdown reduces ER transactivation activity. MCF-7 cells were treated with Ciz1-specific small interfering RNA for 48 hours, transfected with ERE-luciferase for 24 hours, and treated with E2 (10À9 mol/L) for 24 hours, and then ERE-luciferase activity was measured. Inset, knockdown efficiency of Ciz1 by Western blot. Right, Ciz1 overexpression increases ER transactivation activity on the endogenous promoter of pS2. MCF-7 cells were transfected with CMV, Ciz1, and ERE-luciferase for 24 hours and treated with E2 (10À9 mol/L) for 24 hours, and then ERE-luciferase activity was measured. C, Ciz1 and ER form a complex on the ERE. ZR-75 cells were treated with E2 (10À9 mol/L) for 1 hour before the nuclear extracts were made. Bands were supershifted by adding anti-Ciz1 or anti-ER antibody. Arrows, supershift bands. D, Ciz1 induces cell hypersensitivity to estrogen. Top, Ciz1 overexpression induces cyclin D1 expression. ZR-75/pcDNA and ZR-75/Ciz1 cells were treated with E2 (10À9 mol/L) for 24 hours. Cells were pretreated with ICI-182780 for 30 minutes. Bottom, Ciz1 induces cyclin D1 expression even with a low dose of E2. ZR-75/pcDNA and ZR-75/Ciz1 cells were treated with 10À13,10À11,or10À9 mol/L E2 for 24 hours.

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Figure 5. Ciz1 induces a tumorigenic phenotype. A, Ciz1 induces cell proliferation. ZR-75/Ciz1 and ZR-75/pcDNA cells were counted on days 0, 2, 4, and 6. Points, cell number; bars, SD. B, the proliferative advantage of Ciz1 overexpression is blocked by ICI-182780. ZR-75/Ciz1 and ZR-75/pcDNA cells were grown in 5% FBS with or without ICI-182780 and were counted on days 0, 2, 4, and 6. Points, cell number; bars, SD. C, Ciz1 overexpression promotes anchorage- independent colony formation. ZR-75/ pcDNA and ZR-75/Ciz1 cells were plated in soft agar and stimulated with E2 for 21 days. Columns, amount of colonies; bars, SD. D, colony formation advantage of Ciz1 overexpression is blocked by ICI-182780. ZR-75/pcDNA and ZR-75/Ciz1 cells were plated in soft agar with 5% FBS and treated with ICI-182780. Columns, amount of colonies; bars, SD.

growth of Ciz1-overexpressing cells, was inhibited by this agent. consistent with the observation that Ciz1 can promote cell cycle In the absence of ligand-activated ER (due to antiestrogenic treat- progression in other cell types (13). However, because charcoal ment), Ciz1 was unable to induce the transactivation or other stripping can substantially, but not completely, deplete estrogen downstream functions of ER. These findings imply that the effects in serum, the modest ER-promoting activity of Ciz1 might have of Ciz1 on the proliferation and anchorage-independent growth resulted from the residual extremely low levels of estrogen in the of breast tumor cells are dependent on ER. The mechanism by medium. The residual low concentration of estrogen was fully which Ciz1 acts as a coactivator for ER is unclear at the moment. capable of inducing cell growth and anchorage-independent Because Ciz1 is known to interact with the DNA element (18) and growth of Ciz1-expressing cells. In addition, Ciz1-expressing cells because Ciz1 binds to the DNA-binding region of ER, we were extremely sensitive to the femtomolar concentration of hypothesized that the DNA-binding activity of Ciz1 has an inherent estrogen in that their growth and expression of cyclin D1 role in ER-modifying activity. The DNA-binding motif of Ciz1 and increased. Hypersensitivity to estrogen has been observed in its interaction with ER might create a better ER docking site on patients with breast cancer; this phenomenon is typically known the DNA. We previously discovered a glutamic acid–rich region in as adaptive hypersensitivity. In hypersensitive breast cancer the amino acid sequence of Ciz1, a region previously shown to be patients, tumor growth requires premenopausal levels of estrogen important in histone binding (17, 19). Ciz1 may play an important (50-600 pg/mL) initially but later needs only 10 to 15 pg/mL of role in histone modification near the ER target gene chromatin. estrogen (20). This hypersensitivity is believed to be a result of Another notable finding of the present study is that Ciz1 itself adaptive pressure of estrogen depletion over a long period. During is an estrogen-inducible gene and contains ERE motifs. The func- this period of deprivation, cells with particular alterations might tional implication of this finding resides in the potential signal be selected over the remaining tumor cells, leading to better amplification role of Ciz1 in maintaining the duration and strength survival of the hypersensitive cells. In this context, it is important of estrogenic signaling once ER-positive breast cancer cells are to point out that Ciz1 is located on chromosome band 9q34 and momentarily exposed to estrogen. The physiologic relevance of that its amplification has been reported in several cancers (21, 22). this implication was derived by our experiment involving selective However, the relationship of Ciz1 to breast cancer and ER status knockdown expression of Ciz1, which resulted in a substantial remains to be investigated. reduction of the ER responsiveness of breast cancer cells and thus The finding that Ciz1 functions were effectively blocked by the suggested a role of Ciz1 in optimal ER responsiveness. inclusion of a potent ER antagonist in studies using tissue culture Although we observed a profound hyperstimulatory effect of models might be of great interest in therapeutic development Ciz1 in the presence of estrogen, there was a modest but because it suggests that although Ciz1 might confer tumorigenic reproducible effect of Ciz1 alone in promoting the growth of the properties to breast cancer cells, these cells remain sensitive to control cells grown in 5% charcoal-stripped serum. This is treatment modalities targeting the ER. www.aacrjournals.org 11027 Cancer Res 2006; 66: (22). November 15, 2006

Figure 6. Ciz1-overexpressing cells induce tumors in nude mice. A, ZR-75/Ciz1– overexpressing cells have a higher tumor incidence rate than ZR-75/pcDNA cells. ZR-75/pcDNA or ZR-75/Ciz1 cells were injected 2 days after the implantation of the 60-day release estrogen pellets. Mice were monitored for tumor incidence. A, right, tumors from ZR-75/Ciz1 cells growbigger than the ZR-75/pcDNA cells. ZR-75/pcDNA or ZR-75/Ciz1 cells were injected 2 days after the implantation of the 60-day release estrogen pellets. Tumor size was measured every 15 days. Points, tumor volume; bars, SD. B, tumors from ZR-75/Ciz1 cells have a higher level of proliferation then the control tumors. Tumors were dissected, formalin fixed, and paraffin embedded. Sections were stained for PCNA and Ki-67. One representative example. C, tumors from Ciz1-overexpressing cells showmore cyclin D1 expression than the tumors from the control cells. D, working model of the potential role of Ciz1 action in hormone action.

In conclusion, we found that Ciz1 is an estrogen-responsive gene role of Ciz1 in amplifying estrogenic responses and its role in and that Ciz1 protein coregulates ER by enhancing its trans- promoting breast cancer tumorigenesis. activation activity and recruitment to target gene chromatin. Ciz1 induces the hypersensitivity of breast cancer cells to estrogen and induces the expression of ER target gene cyclin D1 at a femtomolar Acknowledgments dose of estrogen, with likely downstream effects on G1 progression Received 6/26/2006; revised 8/18/2006; accepted 9/20/2006. and DNA replication. Moreover, Ciz1 promotes the proliferation, Grant support: NIH grants CA 80066 and CA 65746 (R. Kumar). The costs of publication of this article were defrayed in part by the payment of page anchorage-independent growth, and tumorigenesis of breast charges. This article must therefore be hereby marked advertisement in accordance cancer cells. Taken together, these findings reveal the inherent with 18 U.S.C. Section 1734 solely to indicate this fact.

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